Inhalable TFRC-Targeted Extracellular Vesicles Delivery of siTGF-β1 Alleviates Pulmonary Fibrosis via Dual Inhibition of Ferroptosis and Fibroblast Activation

Background: Idiopathic pulmonary fibrosis (IPF) is a fatal lung disorder marked by excessive extracellular matrix deposition and limited treatment options. Ferroptosis has emerged as a critical driver of epithelial injury and fibrogenesis, while fibroblast activation further accelerates pathological remodeling. The transferrin receptor (TFRC), aberrantly upregulated in both alveolar epithelial cells and fibroblasts during fibrosis, represents a promising target for precision therapy. Methods: An engineered extracellular vesicle (EV) platform was developed using human umbilical cord mesenchymal stem cell-derived vesicles (HucMSC-EVs). By conjugating a T7 peptide for TFRC targeting and encapsulating small interfering RNA against transforming growth factor-beta 1 (siTGF-β 1) through electroporation, a dual-functional nanocomplex (T7-EV/siTGF-β 1) was generated. Its delivery efficiency, molecular effects, and therapeutic outcomes were systematically evaluated in vitro and in bleomycin-induced pulmonary fibrosis mouse models. Results: T7-EV/siTGF-β 1 achieved targeted uptake by epithelial cells and fibroblasts, efficiently silencing TGF-β 1 expression. Treatment significantly inhibited iron accumulation, reactive oxygen species (ROS) generation, and lipid peroxidation, thereby suppressing ferroptosis. Concurrently, the nanocomplex reduced myofibroblast activation, collagen deposition, and fibrotic remodeling, ultimately improving lung histopathology and respiratory function. Importantly, aerosolized administration enabled preferential lung accumulation with minimal off-target distribution and excellent biocompatibility. Conclusion: This study demonstrates that simultaneous inhibition of epithelial ferroptosis and fibroblast activation via TFRC-targeted EV-mediated siRNA delivery effectively mitigates pulmonary fibrosis. T7-EV/siTGF-β 1 thus offers a synergistic and clinically translatable strategy for treating IPF and other fibrotic lung diseases. Plain Language Summary: Alveolar epithelial ferroptosis promotes fibroblast activation through TGF-β 1 signaling.TFRC is upregulated during early-to-mid fibrosis, enabling stage-specific targeted delivery.T7-modified MSC-derived EVs exhibit enhanced TFRC-mediated targeting and siRNA delivery.T7-EV/siTGF-β 1 suppresses ferroptosis and oxidative stress in bleomycin-injured epithelial cells, and inhibits TGF-β 1-mediated fibroblast activation in vitro and vivo. Keywords: extracellular vesicles, T7 peptide, TFRC, ferroptosis, pulmonary fibrosis